Production of sphingoid bases in yeast, pichia ciferrii by metabolic engineering

Abstract

The yeast, Pichia ciferrii has been known to produce large quantities of sphingoid bases, which are secreted into extracellular medium as acetylated bases. These secreted bases consist mostly of tetraacetyl phytosphingosine (TAPS), which is a precursor of sphingolipid synthesis. We have developed an integrative transformation system for improvement of the yield of TAPS and production of other sphingoid bases in the P. ciferrii by metabolic engineering. Ribosomal protein L41 gene of P. ciferrii was cloned using nucleotide sequence homology. To use as a dominant selection marker that confers resistance to the antibiotic cycloheximide, the $56^th$ proline residue of the P. ciferrii L41 gene was changed to glutamine by site directed mutagenesis. The ribosomal DNA (rDNA) repeating unit of P. ciferrii was also cloned to use as target for multicopy gene integration into the chromosome and a locus, which could induce high frequency integration of transforming DNA into the chromosome, was selected. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene encoding a subunit of serine palmitoyltransferase (SPT), which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was cloned from P. ciferrii and its promoter was used for overexpression of LCB2 gene. All transformants transformed with an LCB2 gene expression cassette were observed to produce approximately two times more TAPS than the wild type. In yeast, sphinganine (dihydrosphingosine) is hydroxylated at C-4 by sphinganine hydroxylase (SUR2) to produce phytosphingosine or converted to sphinganine-1-phosphate by sphingoid base kinases (LCB4 and LCB5). The accumulation of sphinganine in Saccharomyces cerevisiae strains defective in conversion of sphinganine, has been identified. Therefore, to produce high level of ...